Plants have the same basic cell cycle as eukaryotes. Not surprising, they also have in common with eukaryotes kinases called cyclin dependent kinases (CDK) that regulate the transitions between different phases of the cell cycle. Arabidopsis, a model plant system used to study the cell cycle have several CDK sub-groups. CDKA is most similar to the cdc2 (CDK1) of mammals and contains the highly conserved Pro Ser Thr Ala Ile Arg Glu (PSTAIRE) amino acid sequence (SEQ ID NO:1) in a region that mediates the interaction with their cyclin partner. Arabidopsis also has a plant-specific group of CDK called CDKB that is not conserved in higher animals. Plants do however lack the mammalian counterpart to the G1 CDKs-CDK4 and CDK6. CDKA has been proposed to be the G1 CDK (that is activated by the plant D-type cyclins), while CDKB has been demonstrated to be predominantly expressed in S-phase and later and, therefore, likely identified as the G2/M specific CDK.
Activation/inactivation of these CDKs drives cells through the cell cycle and also dictates when cells are to exit the cell cycle. Arabidopsis contains up to 49 cyclins grouped into 10 subclasses (see Wang et al., Plant Physiol. 135:1084-1099, 2004). Only the A, B and D-classes appear to play a role in the cell cycle and activate CDKs (Wang et al., Plant Physiol. 135:1084-1099, 2004). CDKA is activated by the D-type cyclins, while CDKB is activated by A- and B-type cyclins.
In animals CDKs are negatively regulated by two families of CDK inhibitors (CKIs). One class, called inhibitor of CDK4 (INK4) is comprised of 4 members (p15, p16, p18 and p19) that bind to and inhibit the G1 CDKs, namely CDK4 and CDK6, from binding the cyclin. The other group of inhibitors is called Kinase Inhibitor Proteins (KIPs) or CIP (CDK Interacting Protein) proteins and they are highly conserved in all animals. The CIP/KIP family predominantly inhibit the cyclin A- and E-CDK2 kinase activity. In plants, putative CKIs have been identified (Wang et al., Nature 386:451-452, 1997; Wang et al., Plant J. 15:501-510, 1998; De Veylder et al., Plant Cell 13:1653-1667, 2001; Jasinski et al., Plant Physiol. 130:1871-1882, 2002) and shown to inhibit purified cyclin/CDK kinase activity in vitro (Wang et al, 1997, supra; Wang et al., Plant J. 24:613-623, 2000; Lui et al., Plant J. 21:379-385, 2000). Expression of plant CKIs showed reduced growth with smaller organs containing larger cells (see Wang et al., 2000, supra; Jasinski et al., J. Cell Sci. 115:973-982, 2001; De Veylder et al., supra; Zhou et al., Plant Cell Rep. 20:967-975, 2002; Zhou et al., Plant J. 35:476-489, 2003; Schnittger et al., Plant Cell 15:303-315, 2003). In Arabidopsis, these CKIs, are called Inhibitors of CDK (ICKs) or KIP related proteins (KRPs). Seven ICK family members have been identified that most closely resemble the CIP/KIP family of CKIs. Each of these ICK/KRP family members has high amino acid sequence identity to p27KIP1 but the identity is limited to the most C-terminal 30 amino acids. To date, no INK related CKIs have been identified in any plant.
Over expression of cyclins or knockouts of CKI illustrate that the well balanced cell cycle engine can easily be perturbed in mammals. This imbalance can ultimately lead to accelerated cell cycles, increased animal size and/or tumor development. Reducing or completely eliminating CKI “activity” results in increased cyclin/CDK kinase activity. This increased activity results in phosphorylation of downstream targets necessary for cell cycle progression and animals ultimately yields cell hyper-proliferation (Coats et al., Science 272:877-880, 1996). Deletion of the p27KIP1 gene in mice results in larger mice due to excessive cyclin/cdk activity that leads to excessive cell proliferation (Fero et al., Cell 85:733-744, 1996; Kiyokawa et al., Cell 85:721-732, 1996; Nakayama et al., Cell 85:707-720, 1996).
Mechanisms exist to suppress expression of various members of the KRP family. Post-transcriptional gene silencing (PTGS) in plants is an RNA-degradation mechanism similar to RNA interference (RNAi) in animals. RNAi results in the specific degradation of double-stranded RNA (dsRNA) into short 21-23 bp dsRNA fragments which ultimately play a role in the degradation of a population of homologous RNAs. In plants, PTGS uses an inverted repeats (IR) strategy to suppress gene expression in many plants species including crop plants such as corn, soy and Canola to name a few. However, IR technology has several drawbacks such as the efficiency of IR sequence, off target gene regulation (Jackson et al., Nature Biotech. 21:635-637, 2003), transient silencing, overall IR stability, and the like. These drawbacks are compounded in the present case by the need to silence more than one gene at a time.
Conventional plant breeding has been the principle driving force for increased crop yields over the past 75 years (J. Femandez-Cornejo, Agriculture Information Bulletin No. (AIB786) 81 pp, February 2004). More recently, transgenic crops have become available that for example have resistance to insect pests and herbicides. However, these transgenic crops do come with a yield penalty (Elmore et al., Agron. J 93:408-412, 2001; Elmore et al., Agron. J. 93:404-407, 2001). To date, no known transgenic crop is commercially available that has an increase in seed size or an increase in crop yield.
There is a need in the art for improved methods of modifying characteristics of certain commercially valuabile crops, including for example, but not limitation, increasing crop yields, increasing seed size, increasing the rate of germination, increasing root mass, and the like. The present invention as described herein meets these and other needs.